Anti-feline herpes virus-1 recombinant antibody and gene fragment coding for said antibody

ABSTRACT

PCT No. PCT/JP93/01724 Sec. 371 Date May 26, 1995 Sec. 102(e) Date May 26, 1995 PCT Filed Nov. 25, 1993 PCT Pub. No. WO94/12661 PCT Pub. Date Sep. 6, 1994An anti-FHV-1 recombinant antibody efficacious for treatment, prevention and diagnosis of feline herpes virus-1 (FHV-1) and a gene fragment useful for preparing the same are provided. A cell producing a mouse monoclonal antibody having an excellent neutralizing activity against FHV-1 was constructed, and a gene fragment coding for V region of said antibody responsible for the specific binding activity against FHV-1 was obtained. Using this gene fragment and a gene fragment coding for the constant region of a feline antibody, a chimeric anti-FHV-1 recombinant antibody is obtained.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a 371 national stage application of PCT/JP93/01724, filed Nov.25, 1993.

TECHNICAL FIELD

The present invention relates to a novel feline monoclonal antibodyusable for diagnosis, treatment and prevention of feline herpes virus-1(FHV-1) infection. More particularly, it relates to a felinizedanti-FHV-1 recombinant antibody wherein a constant region of amouse-type anti-FHV-1 neutralizing monoclonal antibody is replaced witha feline antibody constant region, and a gene fragment encoding saidantibody.

BACKGROUND ART

A cat is such an animal that has been loved by humans as a pet fromancient times and, in modern times, called as Companion species, isbecoming a member of a human society. On the other hand, a cat hashitherto greatly contributed to humans as an experimental animal invarious fields such as medicine, pharmaceutics, animal husbandryveterinary and psychology, and in recent years, the contribution of acat has further increased to be used as an SPF Cat in an effectivenessassay or safety test for drugs. In any case, as a matter of course, morereliable knowledge on feline diseases, especially on infectiousdiseases, is increasingly needed and the establishment of a method fordiagnosis, treatment and prevention of these diseases is required.

Many viral diseases of cats have been known. Among them, an uppertracheal disease caused by FHV-1 is acute and shows a high lethality.For both diseases, there is no specific treating drug and only asymptomatic treatment for preventing a secondary infection by the use ofantibiotics, sulfonamide drugs, etc., and hence, there remains a problemin a conventional method for treatment.

Hitherto, a hiperimmune serum or a serum-derived immunoglobulin havebeen used as a medicament for treatment of viral diseases and have shownsatisfactory results. Nowadays, however, feline serum materials arehardly available due to promotion of Be-Kind-to-Animals thought, andhence, we are in a situation that this treating method cannot be used inspite of desire to use this method. Accordingly, in place of theconventional hiperimmune serum, a monoclonal antibody capable ofneutralizing FHV-1 will possibly greatly contribute to the treatment ofFHV-1 infection.

PRIOR ART

There have been established several neutralizing monoclonal antibodiesagainst FHV-1. However, all the monoclonal antibodies establishedhitherto are antibodies derived from a mouse hybridoma. When theseantibodies are administered to a cat as a medicament, since they are aheterogeneous protein, they will show weaker binding capacity tocomplement or immunocompetent cells having an Fc receptor present inblood than that of homo species (cat) and appear to hardly induce celldamage by "antibody+complement" or antibody-dependent cellularcytotoxicity, cell-mediated cell damage. It is known that, in additionto the action by an antibody alone, these two immune reactions are alsoimportant for prevention of FHV-1 infection and for neutralization ofvirus (Horimoto T. et al., Jpn. J. Vet. Sci. 51, p1025, 1989).Therefore, it is possible that the conventional mouse antibodies cannotexhibit efficacious results in treatment.

Furthermore, it is also possible that the mouse antibodies recognized asa heterogeneous protein cause a side effect such as anaphylaxic shock orserum disease or show a shortened half-life, resulting in a decreasedefficacy in treatment. Accordingly, when administered, the conventionalmouse monoclonal antibodies have never been satisfactory but a felinizedmonoclonal antibody should have been used.

DISCLOSURE OF THE INVENTION

Under the circumstances, the present inventors have established a mousemonoclonal antibody, JH2, which neutralizes FHV-1 viral strain, haveidentified a nucleotide sequence of a gene encoding a variable region (Vregion) of said antibody, and have found a specific amino acid sequencein V region of said antibody which is deeply involved in theneutralization of FHV-1. Then, in order to felinize this mouse typemonoclonal antibody, the present inventors have constructed a vectorexpressing an anti-FHV-1 chimeric antibody having an FHV-1 neutralizingactivity by ligating the antibody V region gene coding for theanti-FHV-1 neutralizing antibody with a gene fragment coding for aconstant region of feline antibody which the present inventors havepreviously found, have expressed said vector, and have successfullyobtained the anti-FHV-1 chimeric antibody. That is, an object of thepresent invention is to provide an anti-FHV-1 felinized chimericantibody which has hitherto never been reported and is prepared byutilizing a genetic engineering technique by replacing the constantregion of the mouse-type antibody with a constant region of a felineantibody, and to provide a gene fragment coding for said antibody usefulfor production of the same. Thus, now it becomes possible to apply ananti-FHV-1 antibody as diagnostic, treating and preventing agents whichis effective against FHV-1 infection without side effects.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1 illustrates results of agarose gel electrophoresis of VH, Vκgenes of the anti-FHV-1 antibody JH2 amplified by PCR.

FIG. 2 shows a base sequence (SEQ ID NO:1) of a VH gene obtained inExample (3) and an amino acid sequence (SEQ ID NO:2) encoded by saidgene.

FIG. 3 shows a base sequence (SEQ ID NO:3) of a Vκ gene obtained inExample (4) and an amino acid sequence (SEQ ID NO:4) encoded by saidgene.

FIG. 4 shows results of homologous comparison of the VH region betweenJH2 (SEQ ID NO:2) and other antibodies (SEQ ID NOS:14-17) at an aminoacid level.

FIG. 5 shows results of homologous comparison of the Vκ region betweenJH2 (SEQ ID NO:4) and other antibodies (SEQ ID NOS:18-21) at an aminoacid level.

FIG. 6 shows a restriction enzyme map of a vector expressing an H chainof anti-FHV-1 chimeric antibody.

FIG. 7 shows a restriction enzyme map of a vector expressing an L chainof anti-FHV-1 chimeric antibody.

FIG. 8 shows a reactivity of the chimeric antibody of the presentinvention with an anti-feline antibody.

FIG. 9 is an illustration showing results of SDS-PAGE of the chimericantibody of the present invention.

FIG. 10 shows a binding of the chimeric antibody of the presentinvention to FHV-1.

FIG. 11 shows effectiveness of the chimeric antibody of the presentinvention for cats compulsorily infected with FHV-1 K1 strain.

FIG. 12 shows effectiveness of the chimeric antibody of the presentinvention for SPF cats compulsorily infected with FHV-1 K1 strain.

BEST MODE FOR PRACTICING THE INVENTION

First, the present inventors have conducted a cell fusion between alymphocyte of mice immunized with FHV-1-K1 viral particles and a mousemyeloma cell in accordance with the conventional manner to prepare ahybridoma. As a result of cloning with an index of viral neutralizingactivity in culture supernatant of hybridomas, the present inventorshave successfully established an antibody-producing cell, JH2, whichproduces a monoclonal antibody being quite excellent in FHV-1neutralizing activity.

It is generally known that a specificity of an antibody against anantigen is based on amino acids in a variable (V) region of an antibody.Thus, the present inventors have studied what amino acids constitute Vregion of JH2. The amino acid sequence was determined by cloning a genecoding for V region of said antibody and studying a nucleotide sequencethereof.

As a result, V region was found to have the amino acid sequence as shownin FIGS. 3 and 4. Generally, there exist about 200 VH, about several 10sD, and 4 JH genes in an H chain gene region of an antibody. On the otherhand, there are about 200 Vκ, and 4 Jκ genes in an L(κ) chain generegion. It is known that, with differentiation of B cells, each one geneis selected from these V(D)J gene fragments and rearranged to form agene coding for an amino acid of a whole variable region. Addition of Nsequence and a Somatic Mutation give a great number of varieties of anantibody variable region. The variable region gene of JH2 of the presentinvention is a unique one selected among such varieties. CDR shown inFigures is an important region for binding with an antigen. Also in caseof JH2, it appears that the amino acid of these six CDR regions bindswith FHV to induce a neutralizing reaction. Especially, "Asp Gly Ala TrpPhe Pro Phe" (corresponding to amino acid residues 126 to 132 of SEQ IDNO:2) in H chain CDR3 was found to be an unique amino acid sequence by ahomologous comparison with variable regions of another antibodies.

Accordingly, the amino acids of H chain CDR3 as well as another CDRregions of H and L chains are considered to be closely related tobinding and neutralizing reactions with FHV-1. This has been firstlyrevealed by isolating the variable region gene of JH2 antibody.Determination of a nucleotide sequence and an amino acid sequence of JH2antibody further enables, based on these sequences, enhancement of anantigen-binding activity and felinization of V region itself. Anantibody or a peptide having such amino acid sequence is conjectured tobe usable for treatment, diagnosis and prevention of FHV-1 infection.

That is, the gene fragment coding for V region of the antibody having aneutralizing activity against FHV-1 as provided according to the presentinvention is a gene fragment having the following characteristics.

A gene fragment coding for VH or a part thereof of the antibody whichspecifically reacts with feline herpes virus-1 (FHV-1) is a genefragment of VH of the antibody wherein the nucleotide sequence codingfor CDR3 of said antibody is a nucleotide sequence coding for thefollowing amino acid sequence:

Asp Gly Ala Trp Phe Pro Phe (amino acid residues 126 to 132 of SEQ IDNO:2).

A preferable gene fragment of VH having a nucleotide sequence coding forsuch sequence includes a gene fragment wherein nucleotide sequencescoding for CDRs 1 to 3 of said antibody are nucleotide sequences eachcoding for the following amino acid sequences:

CDR1: LeuSerThrSerGlyMetGlyAlaGly (amino acids 48 to 56 of SEQ ID NO:2)

CDR2: HisIleTrpTrpAspAspValLysArgTyrAsnProAlaLeuLysSer (amino acids 71to 86 of SEQ ID NO:2)

CDR3: SerGlnIleTyrPheAspTyrAspGlyAlaTrpPheProPhe (amino acids 119 to 132of SEQ ID NO:2)

Furthermore, by way of example, a preferable sequence of the above VHgene fragment includes a nucleotide sequence coding for the amino acidsequence as described in Sequence Listing: SEQ ID NO:2. A specificnucleotide sequence includes, for example, the nucleotide sequencecorresponding to nucleotides 21 to 449 of SEQ ID NO:1.

On the other hand, a gene fragment coding for VL or a part thereof ofthe antibody which specifically reacts with feline herpes virus-1(FHV-1) includes a gene fragment wherein nucleotide sequences coding forCDRs 1 to 3 of said antibody are nucleotide sequences each coding forthe following amino acid sequences:

CDR1: ArgAlaSerGlnSerIleSerAsnAsnLeuHis (amino acids 44 to 54 of SEQ IDNO:4)

CDR2: AlaSerGlnSerIleSerGly (amino acids 71 to 77 of SEQ ID NO:4)

CDR3: GlnGlnSerAsnSerTrpProHisThr (amino acids 109 to 117 of SEQ IDNO:4)

Furthermore, by way of example, a preferable sequence of the above VLgene fragment includes a nucleotide sequence coding for the amino acidsequence as described in Sequence Listing: SEQ ID NO:4. A specificnucleotide sequence includes, for example, the nucleotide sequencecorresponding to nucleotides 23 to 403 of SEQ ID NO:3.

In the meanwhile, it appears to be difficult to administer directly tocats the mouse-type antibody JH2 for treatment of FHV-1 infection inview of reduction of efficacy, side effects, shortened half-life, etc.This is because the antibody itself is a protein heterogeneous to catsderived from mice, and hence, one can foresee that said antibody cannotpossibly bind to a complement or immunocompetent cells having Fcreceptor of cats, and as a result, ADCC or CDC is hardly inducible. Asmentioned above, considering that these activities greatly contribute tothe viral neutralization in FHV infection, the antibody needs to have anFc region derived from cats.

Furthermore, if the antibody exhibits immunogenicity as a heterogenousprotein within the living body, it is possibly cleared away quickly fromthe living body to shorten the half-life or it may provoke side effectssuch as a serum disease. It is said that the immunogenicity of anantibody molecule is localized in the Fc region. In this sense, the Fcregion preferably has an amino acid sequence of an antibody moleculederived from cats as a homogenous protein.

Now, the present inventors have successfully prepared a felinized JH2 byreplacing the antibody constant region of JH2 with that derived fromcats utilizing a genetic engineering technique.

Such felinized antibody, i.e. anti-FHV-1 chimeric antibody, can beprepared by ligating the feline antibody constant region gene; CH geneand CL gene to the downstream (3' site) of the above-mentioned genecoding for VH of anti-FHV-1 antibody and the VL gene provided by thepresent invention to construct a structural gene of antibody H chain andL chain coding for said felinized antibody, i.e. felinized chimericantibody, and expressing said structural gene in a suitable animal cell,etc.

The gene coding for such feline antibody constant region has previouslybeen found by the present inventors (Japanese Patent First PublicationNo. 3-123488, Japanese Patent First Publication No. 3-201986 andJapanese Patent First Publication No. 3-72873). A nucleotide sequence ofthe gene coding for such feline antibody constant region includes, as agene fragment coding for CH, that of a gene fragment coding for theamino acid sequence as described in Sequence Listing: SEQ ID NO:6. Aspecific nucleotide sequence of said gene includes the nucleotidesequence as described in Sequence Listing: SEQ ID NO:5. As a gene codingfor Cκ, a nucleotide sequence of said gene includes that of a genefragment coding for the amino acid sequence as described in SequenceListing: SEQ ID NO:8. A specific nucleotide sequence of said geneincludes the nucleotide sequence as described in Sequence Listing: SEQID NO:7. As a gene coding for Cλ, a nucleotide sequence of said geneincludes that of a gene fragment coding for the amino acid sequence asdescribed in Sequence Listing: SEQ ID NO:10. A specific nucleotidesequence of said gene includes the nucleotide sequence as described inSequence Listing: SEQ ID NO:9.

The gene fragment coding for V region of the antibody having aneutralizing activity against FHV-1 of the present invention, inaddition to the production of the chimeric antibody comprising themouse-derived V region and the cat-derived C region as mentioned above,can also be used for preparing a reshaped antibody in which a frame (FR)region of V region is also replaced with that derived from an antibodyof animals other than mice (in case of the present invention, derivedfrom a feline antibody). There has not yet been totally reported anamino acid sequence of a general V region FR region of a felineantibody. However, a part of such sequence has already been reported(KEHO J. M. et al., Proc. N.A.S. 69, p2052, 1972). Based on these andthe amino acid sequence of the constant region of feline antibody whichthe present inventors have previously found etc., it will be possible toprepare a suitable primer, to clone a gene coding for a feline antibodyV region and to determine an amino acid sequence of said FR region. Areshaped antibody can be prepared basically in accordance with the knowntechnique (for example, Japanese Patent First Publication No.62-296890). The gene fragment of the present invention used in this caseis referred to, as a part of a gene coding for VH chain and VL chain, agene fragment containing at least a nucleotide sequence coding for thefollowing amino acid sequence:

Asp Gly Ala Trp Phe Pro Phe (amino acid residues 126 to 132 of SEQ IDNO:2).

Preferably, nucleic acids coding for CDRs in V region of said reshapedantibody are those coding for the following amino acid sequences for VHchain and VL chain, respectively:

VH chain:

CDR1: LeuSerThrSerGlyMetGlyAlaGly (amino acids 48 to 56 of SEQ ID NO:2)

CDR2: HisIleTrpTrpAspAspValLysArgTyrAsnProAlaLeuLysSer (amino acids 71to 86 of SEQ ID NO:2)

CDR3: SerGlnIleTyrPheAspTyrAspGlyAlaTrpPheProPhe (amino acids 119 to 132of SEQ ID NO:2)

VL chain:

CDR1: ArgAlaSerGlnSerIleSerAsnAsnLeuHis (amino acids 44 to 54 of SEQ IDNO:4)

CDR2: AlaSerGlnSerIleSerGly (amino acids 71 to 77 of SEQ ID NO:4)

CDR3: GlnGlnSerAsnSerTrpProHisThr (amino acids 109 to 117 of SEQ IDNO:4)

In addition, the present inventors have further found previously that,in preparing the reshaped antibody as mentioned above, an antibodyhaving an excellent specificity of the original mouse monoclonalantibody can sometimes be prepared by replacing a portion of FR regionof V region adjacent to CDRs with that derived from mice in addition toreplacement of CDRs in V region with those derived from mice rather thanreplacement of CDRs alone in V region with those derived from mice inaccordance with the conventional technique. That is, by replacing aportion of FR region of V region as well with that derived from mice byreference to the amino acid sequence described in FIG. 2 for VH chainand the amino acid sequence described in FIG. 3 for VL chain, a reshapedantibody can possibly be prepared which is more excellent than thereshaped antibody in which only CDRs are replaced with those derivedfrom mice.

As mentioned above, the structural gene coding for the felinizedanti-FHV-1 recombinant antibody (so called chimeric antibody) isprepared by constructing a V region structural gene for a chimericantibody or a reshaped antibody by the use of the gene coding for thevariable region of the anti-FHV-1 antibody of the present invention andligating this structural gene with the gene coding for the constantregion of feline antibody which the present inventors have previouslyfound. The recombinant antibody of the present invention obtained byligating this structural gene to the downstream of a suitable promotorgene and expressing the resultant in an animal cell, etc. in accordancewith the known technique retained an excellent neutralizing activitylike the mouse-type antibody JH2. When administered to FHV-infectedcats, the recombinant antibody of the present invention alleviated thedisease state without any significant side effect. E. A. Emini et al.have studied a preventive effect of a monoclonal antibody against HIVinfection in chimpanzee. As a result, it was found that a humanizedchimeric antibody could prevent HIV infection (E. A. Emini et al.,Nature, 355, p728, 1992) but the original mouse-type antibody could not(E. A. Emini et al., J. Virol., 64, p3674, 1990). It appears that thisis because the latter could not induce Fc region-dependent ADCC or CDCactivities and showed a shortened half-life.

As for the effectiveness of FH2 antibody in FHV-infected cats found bythe present inventors this time, it appears that the mouse-type antibodyhas a weak effect but the effect can be exerted only by the felinizedantibody like the above-mentioned previous case.

From the above-mentioned point of view, the anti-FHV-1 chimeric antibodyFH2 of the present invention can be a substantial medicament fortreatment and prevention of FHV-1 infection.

The present invention is explained in more detail hereinbelow by meansof Example.

EXAMPLE

(1) Production of hybridoma producing anti-FHV-1 neutralizing monoclonalantibody

A culture supernatant of FL cells (feline lung cells) infected withFHV-1 was precipitated with ammonium sulfate. After dialysis, theprecipitate was resuspended in a phosphate buffer and administeredperitoneally to BALB/c mouse for immunization together with Freund'scomplete adjuvant. After two weeks, mouse lymphocytes and mouse myelomacells (P3U1) were cell-fused by the polyethylene glycol method toprepare hybridomas. Cloning was conducted by an index of a viralneutralizing activity of culture supernatant of hybridomas and therebyfour kinds of FHV-1 neutralizing monoclonal antibody producing cellswere established. The following Table 1 shows a minimum effectiveconcentration of each monoclonal antibody necessary for neutralizing 10TCID₅₀ of FHV-1. Among these monoclonal antibodies, JH2 antibody moststrongly neutralized FHV-1-K1 strain.

                  TABLE 1                                                         ______________________________________                                                           Minimum effective concentration                            Name of antibody                                                                         Class   for neutralization (μg/ml                               ______________________________________                                        JH2        γ1, κ                                                                     0.39                                                       JH3        γ1, κ                                                                     6.3                                                        JH4        γ1, κ                                                                     100                                                        JH7        γ2a, κ                                                                    6.3                                                        ______________________________________                                    

(2) Isolation of variable region gene of anti-FHV-1 antibody (JH2)

Whole RNAs were extracted from 1 to 0.5×10⁷ cells (hybridomas) and mRNAswere purified with Oligo dT column (manufactured by Stratagene, Poly(A)Quick mRNA Purification Kit). A single-stranded cDNA was synthesizedwith reverse transcriptase (Takara; all reagents used for geneticengineering were those manufactured by Takara unless otherwisementioned).

Oligonucleotides having a base sequence at the leader region (MHL34,MKL104) as a 5' primer and those having a base sequence at the J region(MHJ3, MKJ124) as a 3' primer were synthesized. The base sequence ofthese primers are shown hereinbelow.

Primer for amplification of VH chain

MHL341: TCTAGAAGCTTGCCGCCACCATGGGCAGACTTACATTCTCATT (nucleotides 1 to 43of SEQ ID NO:1)

MHJ3 : GAAGATCTGGATCCACTCACCTGCAGAGACAGTGA (SEQ ID NO:11)

Primer for amplification of VK chain

MKL104: GGAATTCAAGCTTGCCGCCACCATGGT(T/A)T(C/T)CTCACCTCAG (SEQ ID NO:12)

MKJ124: CTAGATCTGGATCCACTTACGTTT(T/G)ATTTCCA(A/G)CTT (SEQ ID NO:13)

To 20 ng of cDNA were added each 50 pmol of primers. PCR (PolymeraseChain Reaction) was conducted for 30 cycles, each cycle comprising 94°C. for 1 minute, 55° C. for 1 minute and 72° C. for 1 minute, to amplifythe variable region gene (VH, Vκ) flanked by both primers. FIG. 1 showsan agarose electro-phoresis pattern of the amplified gene fragments. Asize of VH (H chain) and Vκ (L chain) genes were about 400 bp and about400 bp, respectively, and virtually corresponded to the bands asexpected.

(3) Determination of base sequence

A base sequence of each gene fragment amplified in 1 was determined bythe dideoxy method. Each of VH and Vκ gene fragments was cloned intopUC18 and a base sequence thereof was determined by the dideoxy method(manufactured by USB, Sequenase ver. 2).

FIG. 2 shows a base sequence of VH gene of JH2 antibody and an aminoacid sequence coded by said gene. This gene took an open reading frame(ORF) and preserved Cys for forming a domain structure, and hence, wasfound to be suitable for expression. It was also found thatrearrangement occurred at JH3. Then, a homology reference was conductedfor the amino acid sequence coded by this gene. Using GENETYX-CD(Software) as a reference software, a data base of Gene Bank wasreferenced. As a result, antibodies 1 to 5 belonging to VHIII/J606family were found to have highly homologous VH (FIG. 4). In FIG. 4, themark (*) shows an amino acid homologous to that of other VHs and theregion other than this mark represents an amino acid sequence found onlyin JH2. Especially, the underlined portion represents a novel amino acidsequence which has hitherto never been reported and is characteristicregion of JH2 antibody.

FIG. 3 shows a base sequence of Vκ gene of JH2 antibody and an aminoacid sequence coded by said gene. Like in case of H chain, this genetook ORF and preserved Cys for forming a domain structure, and hence,was found to be suitable for expression. It was also found thatrearrangement occurred at Jκ1. FIG. 5 shows a result of a homologyreference. Vκ of JH2 also showed homology with other Vκ.

That is, it appeared that the underlined portion in VH chain is specificfor JH2 antibody and is an important amino acid sequence responsible foran antigen binding activity.

(4) Preparation of anti-FHV-1 chimeric antibody genes

The variable region genes amplified by PCR were ligated to the genecoding for γ chain constant region of feline antibody CB25γ (JapanesePatent First Publication No. 3-201986) or to the gene coding for κ chainconstant region of feline antibody CEK (Japanese Patent FirstPublication No. 3-123488). There were used the chicken β-actin promoter(Japanese Patent Application No. 1-309785) as an expression promoter andneo (Southern P. J. J. Mol. Appl. Genet., 1, 327, 1982) or dhfr gene(Stark, G. R. and Wahl, G. M., Annu. Rev. Biochem., 53, p447, 1984) as aselection marker. FIG. 6 and FIG. 7 show restriction enzyme maps of thechimeric antibody H chain and L chain expression vectors prepared,respectively.

(5) Preparation of stable tranformant

Each 10 μg of the gene coding for the chimeric antibody H chain and L(κ)chain as shown in FIG. 6 and FIG. 7 were digested with PvuI and 2×10⁶mouse myeloma cells P3-X63-Ag8-6.5.3 (ATCC CRL1580) were co-transfectedwith the digested products by using lipofectin (manufactured by BRL).The transfected cells were cultured in 5% FCS/RPMI1640 selection mediumcontaining 0.25×10⁻⁷ M methotrexate (MTX) to select drug-resistantstrains (transformant).

Chimeric antibody producing cells were cloned by a limiting dilutionmethod with an index of feline IgG expressed in culture supernatant toestablish an expression cell, FH2. The properties of this cell wereanalyzed as follows.

(6) Reaction with anti-feline antibody

Culture supernatants of FH2 (chimeric antibody expressing cell) and ofJH2 (mouse monoclonal antibody expressing cell) were added to amicrotiter plate immobilized with an anti-feline antibody (E.Y.LABS.INC)and the reaction was conducted at room temperature for 1 hour. Afterwashing the plate, HRP-anti-feline antibody (E.Y.LABS.INC) was reactedat room temperature for 1 hour. After washing the plate again, color wasdeveloped with TMBZ and an absorbance at 450 nm was measured to analyzethe reactivity with the anti-feline antibody (FIG. 8).

The culture supernatant of FH2 reacted with the anti-feline antibody ina concentration-dependent manner whereas the culture supernatant of JH2which expresses the mouse antibody did not react with the anti-felineantibody. This revealed that the chimeric antibody expressed by FH2cells is a feline antibody.

(7) Identification of chimeric antibody by SDS-PAGE

The chimeric antibody was purified from the culture supernatant withProtein A (manufactured by Bio Rad, MAPS-II). The purified chimericantibody was subjected to 12.5% SDS-PAGE and compared with a feline IgGsample (polyclonal antibody). A molecular weight of the purifiedantibody was determined with a prestained marker manufactured by BioRad.

As shown in FIG. 9, the chimeric antibody detected bands at 5×10⁴ for Hchain and at 2.5×10⁴ for L chain under reduced conditions and a band ataround 1.5×10⁵ under non-reduced conditions. This revealed that H and Lchains form a dimer of H2L2 which has the same form as that of IgGpresent in the cat body.

(8) Reaction with FHV-1 viral particle

Then, an antigen binding activity of the chimeric antibody was studied.The culture supernatant (from FH2 and JH2) was added to a microtiterplate immobilized with FHV-1-K1 (crude product by the ammonium sulfateprecipitation). After washing the plate, it was reacted with eitherHRP-anti-feline antibody or HRP-anti-mouse antibody. Color was developedwith TMBZ and the reactivity with FHV-1 viral particle was studied. Likethe mouse antibody JH2, FH2 antibody specifically reacted with FHV-1-K1.However, the recombinant chimeric antibodies specific for anotherviruses (antibodies having the same feline constant region and differentmouse variable regions) did not reacted (FIG. 10).

(9) Neutralization test against FHV-1 virus

Then, the neutralization activity against FHV-1 of the chimeric antibodywas studied. The culture supernatant (from FH2 and JH2) was reacted with100 TCID₅₀ of FHV-1 virus at 4° C. for 6 hours. Thereto were added0.25×10⁵ CRFK cells and cultured at 37° C. for 2 days. CPE (CellRounding) was observed and a minimum effective concentration forneutralization was determined.

The results are summarized in the following Table 2. As a result, it wasconfirmed that FH2 antibody neutralizes FHV-1 at 1.95 μg/ml.

                  TABLE 2                                                         ______________________________________                                                       Minimum Effective Concentration for                            Antibody       Neutralization (μg/ml)                                      ______________________________________                                        Chimeric antibody (FH2)                                                                      1.95                                                           Mouse antibody (JH2)                                                                         1.25                                                           ______________________________________                                    

(10) Effectiveness and safety of FH2 in cats compulsorily infected withFHV

Cats weighing 1.5 to 3.0 Kg were compulsorily infected with 10⁴ TCID₅₀of FHV-1-K1 strain through nose. At the second day, 30 and 10 mg/Kg ofFH2 was administered via the jugular vein. General clinical conditionssuch as weight, temperature, an amount of taking meals, an amount ofdrinking water and spirits, and respiratory lesions such as tears,conjunctivitis, rhinorrhea, sneeze and cough were observed with thelapse of time and scored as 0 for no symptom, 1 for light symptom, 2 formoderate symptom and 3 for severe symptom. As a result, both groupsadministered with 10 and 30 mg/kg alleviated the symptoms (FIG. 11).Then, SPF cats weighing 1.4 to 2.3 kg were compulsorily infected with10⁴ TCID₅₀ of FHV-1-K1 strain. At the second day, 30 and 10 mg/kg of FH2were administered via the jugular vein and the clinical conditions wereobserved. As a result, the onset of the symptoms were inhibited in thegroup administered with FH2 (FIG. 12). These showed the in vivousefulness of FH2 against FHV infection.

In addition, after administration of FH2, no side effect such asdiarrhea, vomiting or another shock-like symptom was observed, andhence, the safety of FH2 was confirmed.

From the above-mentioned point of view, FH2 antibody can be applied asdiagnostic, treating and preventing agents effective against FHV-1infection with no side effect.

Industrial Applicability of the Invention

The feline monoclonal antibody of the present invention is effectiveagainst feline virus herpes virus-1 infection and is applicable asdiagnostic, treating and preventing agents against said disease.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 21                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 462 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 21..449                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TCTAGAAGCTTGCCGCCACCATGGGCAGACTTACATTCTCATTCCTGCTA50                          MetGlyArgLeuThrPheSerPheLeuLeu                                                1510                                                                          CTGATTGTCCCTGCATATGTCCTGTCCCAGGTTACTCTGAAAGAGTCT98                            LeuIleValProAlaTyrValLeuSerGlnValThrLeuLysGluSer                              152025                                                                        GGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCT146                           GlyProGlyIleLeuGlnProSerGlnThrLeuSerLeuThrCysSer                              303540                                                                        TTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTGCAGGCTGGATT194                           PheSerGlyPheSerLeuSerThrSerGlyMetGlyAlaGlyTrpIle                              455055                                                                        CGTCAGCCATCAGGGAAGGGTCTGGAGTGGCTGGCACACATTTGGTGG242                           ArgGlnProSerGlyLysGlyLeuGluTrpLeuAlaHisIleTrpTrp                              606570                                                                        GATGATGTCAAGCGCTATAACCCAGCCCTGAAGAGCCGACTGACTATC290                           AspAspValLysArgTyrAsnProAlaLeuLysSerArgLeuThrIle                              75808590                                                                      TCCAAGGATACCTCCAGCAGCCAGGTATTCCTCAAGATCGCCAGCGTG338                           SerLysAspThrSerSerSerGlnValPheLeuLysIleAlaSerVal                              95100105                                                                      GACACTGCAGATACTGCCACATATTTTTGTGTTCGATCCCAGATCTAC386                           AspThrAlaAspThrAlaThrTyrPheCysValArgSerGlnIleTyr                              110115120                                                                     TTTGATTACGACGGGGCCTGGTTTCCTTTCTGGGGCCAAGGGACTCTG434                           PheAspTyrAspGlyAlaTrpPheProPheTrpGlyGlnGlyThrLeu                              125130135                                                                     GTCACTGTCTCTGCAGGTGAGTGGATCC462                                               ValThrValSerAla                                                               140                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 143 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetGlyArgLeuThrPheSerPheLeuLeuLeuIleValProAlaTyr                              151015                                                                        ValLeuSerGlnValThrLeuLysGluSerGlyProGlyIleLeuGln                              202530                                                                        ProSerGlnThrLeuSerLeuThrCysSerPheSerGlyPheSerLeu                              354045                                                                        SerThrSerGlyMetGlyAlaGlyTrpIleArgGlnProSerGlyLys                              505560                                                                        GlyLeuGluTrpLeuAlaHisIleTrpTrpAspAspValLysArgTyr                              65707580                                                                      AsnProAlaLeuLysSerArgLeuThrIleSerLysAspThrSerSer                              859095                                                                        SerGlnValPheLeuLysIleAlaSerValAspThrAlaAspThrAla                              100105110                                                                     ThrTyrPheCysValArgSerGlnIleTyrPheAspTyrAspGlyAla                              115120125                                                                     TrpPheProPheTrpGlyGlnGlyThrLeuValThrValSerAla                                 130135140                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 424 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 23..403                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GGAATTCAAGCTTGCCGCCACCATGGTATCCTCACCTCAGTTCCTTGGACTT52                        MetValSerSerProGlnPheLeuGlyLeu                                                145150                                                                        ATGCTTTTTTGGATTTCAGCCTCCAGAGGTGATATTGTGCTAACTCAG100                           MetLeuPheTrpIleSerAlaSerArgGlyAspIleValLeuThrGln                              155160165                                                                     TCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGCGTCAGTCTTTCC148                           SerProAlaThrLeuSerValThrProGlyAspSerValSerLeuSer                              170175180185                                                                  TGCAGGGCCAGCCAAAGTATTAGCAACAACCTACACTGGTATCAACAA196                           CysArgAlaSerGlnSerIleSerAsnAsnLeuHisTrpTyrGlnGln                              190195200                                                                     AAATCACATGAGTCTCCAAGGCTTCTCATCAAGTATGCTTCCCAGTCC244                           LysSerHisGluSerProArgLeuLeuIleLysTyrAlaSerGlnSer                              205210215                                                                     ATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATCAGGGACAGAT292                           IleSerGlyIleProSerArgPheSerGlySerGlySerGlyThrAsp                              220225230                                                                     TTCACTCTCAGTATCAACAGTGTGGAGACTGAAGATTTTGGAATGTAT340                           PheThrLeuSerIleAsnSerValGluThrGluAspPheGlyMetTyr                              235240245                                                                     TTCTGTCAACAGAGTAACAGCTGGCCTCACACGTTCGGTGCTGGGACC388                           PheCysGlnGlnSerAsnSerTrpProHisThrPheGlyAlaGlyThr                              250255260265                                                                  AAGCTGGAGCTGAAACGTAAGTGGATCCAGATCTAG424                                       LysLeuGluLeuLys                                                               270                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 127 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetValSerSerProGlnPheLeuGlyLeuMetLeuPheTrpIleSer                              151015                                                                        AlaSerArgGlyAspIleValLeuThrGlnSerProAlaThrLeuSer                              202530                                                                        ValThrProGlyAspSerValSerLeuSerCysArgAlaSerGlnSer                              354045                                                                        IleSerAsnAsnLeuHisTrpTyrGlnGlnLysSerHisGluSerPro                              505560                                                                        ArgLeuLeuIleLysTyrAlaSerGlnSerIleSerGlyIleProSer                              65707580                                                                      ArgPheSerGlySerGlySerGlyThrAspPheThrLeuSerIleAsn                              859095                                                                        SerValGluThrGluAspPheGlyMetTyrPheCysGlnGlnSerAsn                              100105110                                                                     SerTrpProHisThrPheGlyAlaGlyThrLysLeuGluLeuLys                                 115120125                                                                     (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1005 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..999                                                          (D) OTHER INFORMATION: /function=" k1"                                        /product= "k6"                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ACCACGGCCCCATCGGTGTTCCCACTGGCCCCCAGCTGCGGGACCACA48                            ThrThrAlaProSerValPheProLeuAlaProSerCysGlyThrThr                              130135140                                                                     TCTGGCGCCACCGTGGCCCTGGCCTGCCTGGTGTTAGGCTACTTCCCT96                            SerGlyAlaThrValAlaLeuAlaCysLeuValLeuGlyTyrPhePro                              145150155                                                                     GAGCCGGTGACCGTGTCCTGGAACTCCGGCGCCCTGACCAGCGGTGTG144                           GluProValThrValSerTrpAsnSerGlyAlaLeuThrSerGlyVal                              160165170175                                                                  CACACCTTCCCGGCCGTCCTGCAGGCCTCGGGGCTGTACTCTCTCAGC192                           HisThrPheProAlaValLeuGlnAlaSerGlyLeuTyrSerLeuSer                              180185190                                                                     AGCATGGTGACAGTGCCCTCCAGCAGGTGGCTCAGTGACACCTTCACC240                           SerMetValThrValProSerSerArgTrpLeuSerAspThrPheThr                              195200205                                                                     TGCAACGTGGCCCACCCGCCCAGCAACACCAAGGTGGACAAGACCGTG288                           CysAsnValAlaHisProProSerAsnThrLysValAspLysThrVal                              210215220                                                                     CGCAAAACAGACCACCCACCGGGACCCAAACCCTGCGACTGTCCCAAA336                           ArgLysThrAspHisProProGlyProLysProCysAspCysProLys                              225230235                                                                     TGCCCACCCCCTGAGATGCTTGGAGGACCGTCCATCTTCATCTTCCCC384                           CysProProProGluMetLeuGlyGlyProSerIlePheIlePhePro                              240245250255                                                                  CCAAAACCCAAGGACACCCTCTCGATTTCCCGGACGCCCGAGGTCACA432                           ProLysProLysAspThrLeuSerIleSerArgThrProGluValThr                              260265270                                                                     TGCTTGGTGGTGGACTTGGGCCCAGATGACTCCGATGTCCAGATCACA480                           CysLeuValValAspLeuGlyProAspAspSerAspValGlnIleThr                              275280285                                                                     TGGTTTGTGGATAACACCCAGGTGTACACAGCCAAGACGAGTCCGCGT528                           TrpPheValAspAsnThrGlnValTyrThrAlaLysThrSerProArg                              290295300                                                                     GAGGAGCAGTTCAACAGCACCTACCGTGTGGTCAGTGTCCTCCCCATC576                           GluGluGlnPheAsnSerThrTyrArgValValSerValLeuProIle                              305310315                                                                     CTACACCAGGACTGGCTCAAGGGGAAGGAGTTCAAGTGCAAGGTCAAC624                           LeuHisGlnAspTrpLeuLysGlyLysGluPheLysCysLysValAsn                              320325330335                                                                  AGCAAATCCCTCCCCTCCCCCATCGAGAGGACCATCTCCAAGGCCAAA672                           SerLysSerLeuProSerProIleGluArgThrIleSerLysAlaLys                              340345350                                                                     GGACAGCCCCACGAGCCCCAGGTGTACGTCCTGCCTCCAGCCCAGGAG720                           GlyGlnProHisGluProGlnValTyrValLeuProProAlaGlnGlu                              355360365                                                                     GAGCTCAGCAGGAACAAAGTCAGTGTGACCTGCCTGATCAAAAGCTTC768                           GluLeuSerArgAsnLysValSerValThrCysLeuIleLysSerPhe                              370375380                                                                     CACCCGCCTGACATTGCCGTCGAGTGGGAGATCACCGGACAGCCGGAG816                           HisProProAspIleAlaValGluTrpGluIleThrGlyGlnProGlu                              385390395                                                                     CCAGAGAACAACTACCGGACGACCCCGCCCCAGCTGGACAGCGACGGG864                           ProGluAsnAsnTyrArgThrThrProProGlnLeuAspSerAspGly                              400405410415                                                                  ACCTACTTCGTGTACAGCAAGCTCTCGGTGGACAGGTCCCACTGGCAG912                           ThrTyrPheValTyrSerLysLeuSerValAspArgSerHisTrpGln                              420425430                                                                     AGGGGAAACACCTACACCTGCTCGGTGTCACACGAAGCTCTGCACAGC960                           ArgGlyAsnThrTyrThrCysSerValSerHisGluAlaLeuHisSer                              435440445                                                                     CACCACACACAGAAATCCCTCACCCAGTCTCCGGGTAAATGAGCA1005                             HisHisThrGlnLysSerLeuThrGlnSerProGlyLys                                       450455460                                                                     (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 333 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ThrThrAlaProSerValPheProLeuAlaProSerCysGlyThrThr                              151015                                                                        SerGlyAlaThrValAlaLeuAlaCysLeuValLeuGlyTyrPhePro                              202530                                                                        GluProValThrValSerTrpAsnSerGlyAlaLeuThrSerGlyVal                              354045                                                                        HisThrPheProAlaValLeuGlnAlaSerGlyLeuTyrSerLeuSer                              505560                                                                        SerMetValThrValProSerSerArgTrpLeuSerAspThrPheThr                              65707580                                                                      CysAsnValAlaHisProProSerAsnThrLysValAspLysThrVal                              859095                                                                        ArgLysThrAspHisProProGlyProLysProCysAspCysProLys                              100105110                                                                     CysProProProGluMetLeuGlyGlyProSerIlePheIlePhePro                              115120125                                                                     ProLysProLysAspThrLeuSerIleSerArgThrProGluValThr                              130135140                                                                     CysLeuValValAspLeuGlyProAspAspSerAspValGlnIleThr                              145150155160                                                                  TrpPheValAspAsnThrGlnValTyrThrAlaLysThrSerProArg                              165170175                                                                     GluGluGlnPheAsnSerThrTyrArgValValSerValLeuProIle                              180185190                                                                     LeuHisGlnAspTrpLeuLysGlyLysGluPheLysCysLysValAsn                              195200205                                                                     SerLysSerLeuProSerProIleGluArgThrIleSerLysAlaLys                              210215220                                                                     GlyGlnProHisGluProGlnValTyrValLeuProProAlaGlnGlu                              225230235240                                                                  GluLeuSerArgAsnLysValSerValThrCysLeuIleLysSerPhe                              245250255                                                                     HisProProAspIleAlaValGluTrpGluIleThrGlyGlnProGlu                              260265270                                                                     ProGluAsnAsnTyrArgThrThrProProGlnLeuAspSerAspGly                              275280285                                                                     ThrTyrPheValTyrSerLysLeuSerValAspArgSerHisTrpGln                              290295300                                                                     ArgGlyAsnThrTyrThrCysSerValSerHisGluAlaLeuHisSer                              305310315320                                                                  HisHisThrGlnLysSerLeuThrGlnSerProGlyLys                                       325330                                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 337 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..327                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AGTGATGCTCAGCCATCTGTCTTTCTCTTCCAACCATCTCTGGACGAG48                            SerAspAlaGlnProSerValPheLeuPheGlnProSerLeuAspGlu                              335340345                                                                     TTACATACAGGAAGTGCCTCTATCGTGTGCATATTGAATGACTTCTAC96                            LeuHisThrGlySerAlaSerIleValCysIleLeuAsnAspPheTyr                              350355360365                                                                  CCCAAAGAGGTCAATGTCAAGTGGAAAGTGGATGGCGTAGTCCAAACA144                           ProLysGluValAsnValLysTrpLysValAspGlyValValGlnThr                              370375380                                                                     AAGGCATCCAAGGAGAGCACCACAGAGCAGAACAGCAAGGACAGCACC192                           LysAlaSerLysGluSerThrThrGluGlnAsnSerLysAspSerThr                              385390395                                                                     TACAGCCTCAGCAGCACCCTGACGATGTCCAGGACGGAGTACCAAAGT240                           TyrSerLeuSerSerThrLeuThrMetSerArgThrGluTyrGlnSer                              400405410                                                                     CATGAAAAGTTCTCCTGCGAGGTCACTCACAAGAGCCTGGCCTCCACC288                           HisGluLysPheSerCysGluValThrHisLysSerLeuAlaSerThr                              415420425                                                                     CTCGTCAAGAGCTTCAACAGGAGCGAGTGTCAGAGAGAGTAGCCTAGCA337                          LeuValLysSerPheAsnArgSerGluCysGlnArgGlu                                       430435440                                                                     (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 109 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       SerAspAlaGlnProSerValPheLeuPheGlnProSerLeuAspGlu                              151015                                                                        LeuHisThrGlySerAlaSerIleValCysIleLeuAsnAspPheTyr                              202530                                                                        ProLysGluValAsnValLysTrpLysValAspGlyValValGlnThr                              354045                                                                        LysAlaSerLysGluSerThrThrGluGlnAsnSerLysAspSerThr                              505560                                                                        TyrSerLeuSerSerThrLeuThrMetSerArgThrGluTyrGlnSer                              65707580                                                                      HisGluLysPheSerCysGluValThrHisLysSerLeuAlaSerThr                              859095                                                                        LeuValLysSerPheAsnArgSerGluCysGlnArgGlu                                       100105                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 318 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..315                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CAGCCCAAGTCGGCCCCCTCGGTCACACTCTTCCCACCCTCCAGTGAG48                            GlnProLysSerAlaProSerValThrLeuPheProProSerSerGlu                              110115120125                                                                  GAGCTCAGCGCAAACAAGGCCACCCTGGTGTGTCTCGTCAGTGACTTC96                            GluLeuSerAlaAsnLysAlaThrLeuValCysLeuValSerAspPhe                              130135140                                                                     TACCCCAGCGGCTTGACGGTGGCCTGGAAGGAAGATGGCACCCCCATC144                           TyrProSerGlyLeuThrValAlaTrpLysGluAspGlyThrProIle                              145150155                                                                     ACCAAGGGCGTGGAGACCACCAAGCCCTCCAGACAGAGCAACAACAAG192                           ThrLysGlyValGluThrThrLysProSerArgGlnSerAsnAsnLys                              160165170                                                                     TACGCGGCCAGCAGCTACCTGAGCCTGTCACCGAACGAGTGGAAATCT240                           TyrAlaAlaSerSerTyrLeuSerLeuSerProAsnGluTrpLysSer                              175180185                                                                     CACAGCAGATACACCTGCCAGGTCACGCACGAGGGGAGCACTGTGGAG288                           HisSerArgTyrThrCysGlnValThrHisGluGlySerThrValGlu                              190195200205                                                                  AAGAGTGTGGTCCCTGCAGAGTGCCCTTAG318                                             LysSerValValProAlaGluCysPro                                                   210                                                                           (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 105 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GlnProLysSerAlaProSerValThrLeuPheProProSerSerGlu                              151015                                                                        GluLeuSerAlaAsnLysAlaThrLeuValCysLeuValSerAspPhe                              202530                                                                        TyrProSerGlyLeuThrValAlaTrpLysGluAspGlyThrProIle                              354045                                                                        ThrLysGlyValGluThrThrLysProSerArgGlnSerAsnAsnLys                              505560                                                                        TyrAlaAlaSerSerTyrLeuSerLeuSerProAsnGluTrpLysSer                              65707580                                                                      HisSerArgTyrThrCysGlnValThrHisGluGlySerThrValGlu                              859095                                                                        LysSerValValProAlaGluCysPro                                                   100105                                                                        (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GAAGATCTGGATCCACTCACCTGCAGAGACAGTGA35                                         (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GGAATTCAAGCTTGCCGCCACCATGGTWTYCTCACCTCAG40                                    (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CTAGATCTGGATCCACTTACGTTTKATTTCCARCTT36                                        (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 122 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      AspValGlnHisIleSerPheGlnValThrLeuLysGluSerGlyPro                              151015                                                                        GlyIleLeuGlnProSerGlnThrLeuSerLeuThrCysSerPheSer                              202530                                                                        GlyPheSerLeuSerThrSerGlyMetGlyValSerTrpIleArgGln                              354045                                                                        ProSerGlyLysGlyLeuGluTrpLeuAlaHisIleTyrTrpAspAsp                              505560                                                                        AspLysArgTyrAsnProSerLeuLysSerArgLeuThrIleSerLys                              65707580                                                                      AspThrSerArgAsnGlnValPheLeuLysIleThrSerValAspThr                              859095                                                                        AlaAspThrAlaThrTyrTyrCysAlaArgArgGluGlyGlyArgSer                              100105110                                                                     TyrPheAspTyrTrpGlyGlnGlyThrThr                                                115120                                                                        (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 108 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ProGlyIleLeuGlnProSerGlnThrLeuSerLeuThrCysSerPhe                              151015                                                                        SerGlyPheSerLeuSerThrSerGlyMetGlyValGlyTrpIleArg                              202530                                                                        GlnProSerGlyLysGlyLeuGluTrpLeuAlaHisIleTrpTrpAsp                              354045                                                                        AspAspLysArgTyrAsnProAlaLeuLysSerArgLeuThrIleSer                              505560                                                                        LysAspThrSerSerAsnGlnValPheLeuLysIleAlaSerValAsp                              65707580                                                                      ThrAlaAspThrSerThrTyrTyrCysAlaArgAspTrpAspTyrPhe                              859095                                                                        AspTyrTrpGlyGlnGlyThrThrLeuThrValSer                                          100105                                                                        (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 114 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GlyIleLeuGlnProSerGlnThrLeuSerLeuThrCysSerPheSer                              151015                                                                        GlyPheSerLeuSerThrSerGlyMetGlyValGlyTrpIleArgGln                              202530                                                                        ProSerGlyLysGlyLeuGluTrpLeuAlaHisIleTrpTrpAspAsp                              354045                                                                        AspLysArgTyrAsnProAlaLeuLysSerArgLeuThrIleSerLys                              505560                                                                        AspThrSerSerAsnGlnValSerLeuLysIleAlaSerValAspThr                              65707580                                                                      AlaAspThrAlaThrTyrTyrCysAlaArgThrTyrTyrTyrGlySer                              859095                                                                        SerHisTrpTyrPheAspValTrpGlyAlaGlyThrThrValThrVal                              100105110                                                                     SerSer                                                                        (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 108 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      ProGlyIleLeuGlnProSerGlnThrLeuSerLeuThrCysSerPhe                              151015                                                                        SerGlyPheSerLeuSerThrSerGlyMetGlySerTrpIleArgGln                              202530                                                                        ProSerGlyLysGlyLeuGluTrpLeuAlaHisIleTyrTrpAspAsp                              354045                                                                        AspLysArgTyrAsnProSerLeuLysSerArgLeuThrIleSerLys                              505560                                                                        AspThrSerArgAsnGlnValPheLeuLysIleThrSerValAspThr                              65707580                                                                      AlaAspThrAlaThrTyrTyrCysAlaArgSerTyrGlyAsnGlyAsp                              859095                                                                        TyrTyrAlaMetAspTyrTrpGlyGlnGlyThrSer                                          100105                                                                        (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 127 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      MetValPheThrProGlnIleLeuGlyLeuMetLeuPheTrpIleSer                              151015                                                                        AlaSerArgGlyAspIleValLeuThrGlnSerProAlaThrLeuSer                              202530                                                                        ValThrProArgAspSerValSerLeuSerCysArgAlaSerGlnSer                              354045                                                                        IleSerAsnAsnLeuHisTrpTyrGlnGlnLysSerHisGluSerPro                              505560                                                                        ArgLeuLeuIleLysTyrAlaSerGlnSerIleSerGlyIleProSer                              65707580                                                                      ArgPheSerGlySerGlySerGlyThrAspPheThrLeuSerIleAsn                              859095                                                                        SerValGluThrGluAspPheGlyMetTyrPheCysGlnGlnSerAsn                              100105110                                                                     SerTrpProLeuThrPheGlyAlaGlyThrLysLeuGluLeuLys                                 115120125                                                                     (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 100 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      AspIleValLeuThrGlnSerProAlaThrLeuSerValThrProGly                              151015                                                                        AspSerValSerLeuSerCysArgAlaSerGlnSerIleSerAsnAsn                              202530                                                                        LeuHisTrpTyrGlnGlnLysSerHisGluSerProArgLeuLeuIle                              354045                                                                        LysTyrAlaSerGlnSerIleSerGlyIleProSerArgPheSerGly                              505560                                                                        SerGlySerGlyThrAspPheThrLeuSerIleAsnSerValGluThr                              65707580                                                                      GluAspPheGlyMetTyrPheCysGlnGlnSerAsnSerTrpProHis                              859095                                                                        ThrPheGlySer                                                                  100                                                                           (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 107 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      AspIleValLeuThrGlnSerProAlaThrLeuSerValThrProGly                              151015                                                                        AsnSerValSerLeuSerCysArgAlaSerGlnSerIleGlyAsnAsn                              202530                                                                        LeuHisTrpTyrGlnGlnLysSerHisGluSerProArgLeuLeuIle                              354045                                                                        LysTyrAlaSerGlnSerIleSerGlyIleProSerArgPheSerGly                              505560                                                                        SerGlySerGlyThrAspPheThrLeuSerIleAsnSerValGluThr                              65707580                                                                      GluAspPheGlyMetTyrPheCysGlnGlnSerAsnSerTrpProTyr                              859095                                                                        ThrPheGlyGlyGlyThrLysLeuGluIleLys                                             100105                                                                        (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 103 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      IleValLeuThrGlnSerProAlaThrLeuSerValThrProGlyAsp                              151015                                                                        SerValLeuSerCysArgAlaSerGlnSerIleSerAsnAsnLeuHis                              202530                                                                        TrpTyrGlnGlnLysSerHisGluSerProArgLeuLeuIleAsnTyr                              354045                                                                        AlaSerGlnSerIleSerIleProSerArgPheSerGlySerGlySer                              505560                                                                        GlyThrAspPheThrLeuIleIleAsnAsnValGluThrGluAspPhe                              65707580                                                                      GlyMetTyrPheCysGlnGlnSerAsnSerTrpProLeuThrPheAla                              859095                                                                        AlaGlyThrLysLeuGluLeu                                                         100                                                                           __________________________________________________________________________

We claim:
 1. A felinized chimeric antibody having a heavy chain variableregion (VH) and a light chain variable region (VL) obtained from amurine antibody, which specifically reacts with feline herpes virus-1(FHV-1) and has a neutralizing activity against FHV-1, wherein the aminoacid sequence of one of the complementarity determining regions (CDRs)of heavy chain variable region (VH) has the amino acid sequence Asp GlyAla Trp Phe Pro Phe (corresponding to amino acid residues 126 to 132 ofSEQ ID NO:2) and the amino acid sequences of the constant (C) regions ofthe heavy (H) and light (L) chains are those obtained from a felineantibody.
 2. The antibody of claim 1 wherein the CDRs in said VH and VLregions have the following amino acid sequences: VH:CDR1:LeuSerThrSerGlyMetGlyAlaGly corresponding to amino acids 48 to 56 of SEQID NO:2; CDR2: HisIleTrpTrpAspAspValLysArgTyrAsnProAlaLeuLysSercorresponding to amino acids 71 to 86 of SEQ ID NO:2; CDR3:SerGlnIleTyrPheAspTyrAspGlyAlaTrpPheProPhe corresponding to amino acids119 to 132 of SEQ ID NO:2; VL: CDR1: ArgAlaSerGlnSerIleSerAsnAsnLeuHiscorresponding to amino acids 44 to 54 of SEQ ID NO:4; CDR2:AlaSerGlnSerIleSerGly corresponding to amino acids 71 to 77 of SEQ IDNO:4; and CDR3: GlnGlnSerAsnSerTrpProHisThr corresponding to amino acids109 to 117 of SEQ ID NO:4.
 3. The antibody of claim 1 wherein said VHand VL regions have the amino acid sequences of amino acid residues 20to 143 in SEQ ID NO:2 and of amino acid residues 21 to 127 in SEQ IDNO:4, respectively.
 4. The antibody of claim 1 wherein the amino acidsequence of the H chain constant region of said antibody is that of SEQID NO:6 and the amino acid sequence of the L chain constant region ofsaid antibody is that of SEQ ID NO:8 or of SEQ ID NO:10.
 5. A genefragment coding for a heavy chain variable region (VH) or a part thereofof an antibody which specifically reacts with feline herpes virus-1(FHV-1) in which the nucleotide sequence coding for complementaritydetermining region 3 (CDR3) comprises a nucleotide sequence coding forthe amino acid sequence Asp Gly Ala Trp Phe Pro Phe corresponding toamino acid residues 126 to 132 of SEQ ID NO:2.
 6. The gene fragment ofclaim 5 wherein the nucleotide sequences coding for CDRs 1 to 3 arenucleotide sequences coding for the following amino acid sequences,respectively:CDR1 : LeuSerThrSerGlyMetGlyAlaGly corresponding to aminoacids 48 to 56 of SEQ ID NO:2. CDR2 :HisIleTrpTrpAspAspValLysArgTyrAsnProAlaLeuLysSer corresponding to aminoacids 71 to 86 of SEQ ID NO:2. CDR3:SerGlnIleTyrPheAspTyrAspGlyAlaTrpPheProPhe corresponding to amino acids119 to 132 of SEQ ID NO:2.
 7. The gene fragment of claim 5 wherein thenucleotide sequence coding for VH of the antibody is a nucleotidesequence coding for the amino acid sequence of SEQ ID NO:2.
 8. The genefragment of claim 5 wherein the nucleotide sequence coding for VH of theantibody is the corresponding to nucleotides 21 to 449 of SEQ ID NO:1.9. A gene fragment coding for a light chain variable region (VL) of anantibody wherein the nucleotide sequences coding for CDRs 1 to 3 arenucleotide sequences coding for the following amino acid sequences,respectively:CDR1: ArgAlaSerGlnSerIleSerAsnAsnLeuHis corresponding toamino acids 44 to 54 of SEQ ID NO:4; CDR2: AlaSerGlnSerIleSerGlycorresponding to amino acids 71 to 77 of SEQ ID NO:4 and CDR3:GlnGlnSerAsnSerTrpProHisThr corresponding to amino acids 109 to 117 ofSEQ ID NO:4.
 10. The gene fragment of claim 9 wherein the nucleotidesequence coding for VL of the antibody is a nucleotide sequence codingfor the amino acid sequence of SEQ ID NO:4.
 11. The gene fragment ofclaim 9 wherein the nucleotide sequence coding for VL of the antibody isthe nucleotide corresponding to nucleotides 23 to 403 of SEQ ID NO:3.12. A recombinant gene fragment coding for heavy (H) chain of a felinechimeric antibody which specifically reacts with feline herpes virus-1(FHV-1), said gene fragment comprising the gene fragment of claim 5 tothe downstream (3' site) of which a gene fragment coding for H chainconstant region of a feline antibody is bound.
 13. The recombinant genefragment of claim 12 wherein said gene fragment coding for H chainconstant region of a feline antibody has a nucleotide sequence codingfor the amino acid sequence of SEQ ID NO:6.
 14. A recombinant genefragment coding for light (L) chain of a feline chimeric antibody whichspecifically reacts with feline herpes virus-1 (FHV-1), said genefragment comprising the gene fragment of claim 9 to the downstream (3'site) of which a gene fragment coding for κ chain constant region of afeline antibody is bound.
 15. The recombinant gene fragment of claim 14wherein said gene fragment coding for κ chain constant region of afeline antibody has a nucleotide sequence coding for the amino acidsequence of SEQ ID NO:8.
 16. A recombinant gene fragment coding for Lchain of a feline chimeric antibody which specifically reacts withfeline herpes virus-1 (FHV-1), said gene fragment comprising the genefragment of claim 9 to the downstream (3' site) of which a gene fragmentcoding for λ chain constant region of a feline antibody is bound. 17.The recombinant gene fragment of claim 16 wherein said gene fragmentcoding for λ chain constant region of a feline antibody has a nucleotidesequence coding for the amino acid sequence of SEQ ID NO:10.
 18. Amethod of treating FHV-1-infected cats, comprising the step ofadministering a pharmaceutical composition, comprising the felinizedchimeric antibody according to claim 2 and a pharmaceutically acceptableexcipient, to a cat in need thereof.